Nucleic Acids Research, 2001, Vol. 29, No. 14 3108-3115
© 2001 Oxford University Press
Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing
Department of Biochemistry and Molecular Biology, School of Pharmacy, Diagonal 643, University of Barcelona, 08028 Barcelona, Spain, 1IIBB Consejo Superior de Investigaciones Científicas, 08034 Barcelona, Spain and 2Institut für Molekularbiologie, Freie Universität Berlin, D-14195 Berlin, Germany
Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which may lead to the synthesis of two proteins. Generation of the three COT transcripts in rat does not depend on sex, development, fat feeding, the inclusion of the peroxisome proliferator diethylhexyl phthalate in the diet or hyperinsulinemia. In addition, trans-splicing was not detected in COT of other mammals, such as human, pig, cow and mouse, or in Cos7 cells from monkey. Rat COT exon 2 contains two purine-rich sequences. Mutation of the rat COT exon 2 upstream box does not affect the trans-splicing in vitro between two truncated constructs containing exon 2 and its adjacent intron boundaries. In contrast, mutation of the downstream box from the rat sequence (GAAGAAG) to a random sequence or the sequence observed in the other mammals (AAAAAAA) decreased trans-splicing in vitro. In contrast, mutation of the AAAAAAA box of human COT exon 2 to GAAGAAG increases trans-splicing. Heterologous reactions between COT exon 2 from rat and human do not produce trans-splicing. HeLa cells transfected with minigenes of rat COT sequences produced cis- and trans-spliced bands. Mutation of the GAAGAAG box to AAAAAAA abolished trans-splicing and decreased cis-splicing in vivo. We conclude that GAAGAAG is an exonic splicing enhancer that could induce natural trans-splicing in rat COT.
* To whom correspondence should be addressed. Tel: +34 93 402 4523; Fax: +34 93 402 4520; Email: hegardt{at}farmacia.far.ub.es The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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